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A double recombinant protein of Vibrio vulnificus eel and Edwardsiella tarda and its preparation method

A technology of Vibrio vulnificus and recombinant protein, which is applied in the direction of chemical instruments and methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of cumbersome operation and large damage to fish body, so as to simplify the immunization steps and reduce the risk of fish body damage. damage, good immune effect

Active Publication Date: 2020-04-21
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, there have been researches on whole-bacteria inactivated vaccines and subunit vaccines of Vibrio vulnificus and Edwardsiella tarda, but to apply these research results to immunize eels against multiple pathogens requires multiple injections of eels , greater damage to the fish body, and the operation is cumbersome

Method used

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  • A double recombinant protein of Vibrio vulnificus eel and Edwardsiella tarda and its preparation method
  • A double recombinant protein of Vibrio vulnificus eel and Edwardsiella tarda and its preparation method
  • A double recombinant protein of Vibrio vulnificus eel and Edwardsiella tarda and its preparation method

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Embodiment 1

[0037] In this example, the double recombinant protein of Vibrio vulnificus eel and Edwardsiella tarda of the present invention was carried out.

[0038] Preparation of (OMP-Vibrio-Edwa)

[0039] (1) Selected DNA fragments with good immunogenicity and hydrophilicity in the outer membrane region of the outer membrane protein ompU of Vibrio vulnificus and ompA gene of Edwardsiella tarda, and connected the two selected fragments with a linker Get up, design and amplify the upstream and downstream primers of OmpU gene partial fragment respectively: P1:5' CCG GAA TTC TAC GCA GGT CTA GGCGGC AAG T 3' (SEQ ID NO 03, 31bp, introduce EcoRI site); P2:5 'ACC CGA GCC ACC ACC GCCCGAGCC TAT ACG AGC GTA GCC AGC ACC GCC AAC T 3' (SEQ ID NO 04, 52bp); the upstream and downstream primers for amplifying part of the OmpA gene were: P3: 5' TCG GGC GGT GGC GGC TCG GGT GGC GGA TCA GTATGG CGT TCT GAT ATC CAC GG3'(SEQ ID NO 05, 53bp); P4:5'CGC GTC GAC CTG CGG CTGAGA AAC TTC TTC TT 3'(SEQ ID NO 06, 32b...

Embodiment 2

[0059] (1) PBS blank control, Vibrio vulnificus and Edwardsiella tarda dual inactivated bacterial liquid and the Vibrio vulnificus of the present invention obtained in Example 1 and Edwardsiella tarda dual recombinant protein were injected intraperitoneally respectively Three groups of eels (50g / tail) were immunized, and each group of eels corresponded to the PBS control group (PBS Group), the double inactivated bacteria immunization group (Bivalent FKC Group) and the double expression outer membrane protein immunization group (Bivalent OMP Group) , wherein the Vibrio vulnificus of the present invention and Edwardsiella tarda double recombinant protein were prepared into 1 mg / mL with 0.01M PBS (pH=7.4) before the test, and it was fully mixed with an equivalent amount of Freund's incomplete adjuvant Mix well, and the final concentration is 0.5mg / mL; Vibrio vulnificus and Edwardsiella tarda were cultured in tryptone soy broth (TSB) at 28°C and 32°C for 24h, respectively, and then...

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Abstract

The invention discloses an eel Vibrio vulnificus / Edwardsiella tarda duplex recombinant protein and a preparation method thereof. The duplex recombinant protein comprises a Vibrio vulnificus outer membrane protein OmpU extramembrane part and an Edwardsiella tarda outer membrane protein OmpA extramembrane part which are connected together, wherein the Vibrio vulnificus outer membrane protein OmpU extramembrane part and Edwardsiella tarda outer membrane protein OmpA extramembrane part respectively comprise 238th-456th amino acid sequences and 467th-695th amino acid sequences in SEQ ID NO.01. The Vibrio vulnificus outer membrane protein OmpU extramembrane part and Edwardsiella tarda outer membrane protein OmpA extramembrane part are connected together by a gene engineering means to establish the duplex recombinant protein. After being used for immunizing the eel, the protein can simultaneously resist the infection of Vibrio vulnificus and Edwardsiella tarda. Compared with the duplex inactivated vaccine, the duplex recombinant protein has higher relative immunity protection effect and lower toxic and side effects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a dual recombinant protein of Vibrio vulnificus eel and Edwardsiella tarda and a preparation method thereof. Background technique [0002] In the past 20 years, fulminant septicemia has occurred in freshwater cultured fish in my country, which has brought immeasurable economic losses to the aquaculture industry. Studies have shown that the main pathogenic bacteria of the disease are Vibrio vulnificus and Edwardsiella tarda. Vibrio vulnificus (Aeromonas hydrophila) and Edwardsiella tarda (Edwardsiella tarda) are important pathogenic bacteria of eel, which can cause eel hemorrhagic sepsis, liver and kidney enlargement, gill rot, tail rot, etc. Strong diseases can occur in artificially cultured American eels, European eels and Japanese eels. The aquaculture industry has used chemical drugs to treat the disease for a long time. Due to drug resistance and other reasons, the a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/195C07K14/24C07K2319/00C12N15/62
Inventor 郭松林胡琳琳冯建军林鹏
Owner JIMEI UNIV
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